Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device.

The emerging monkeypox virus (MPXV) has raised global health concern, thereby highlighting the need for rapid, sensitive, and easy-to-use diagnostics. Here, we develop a single-step CRISPR-based diagnostic platform, termed SCOPE (Streamlined CRISPR On Pod Evaluation platform), for field-deployable ultrasensitive detection of MPXV in resource-limited settings. The viral nucleic acids are rapidly released from the rash fluid swab, oral swab, saliva, and urine samples in 2 min via a streamlined viral lysis protocol, followed by a 10-min single-step recombinase polymerase amplification (RPA)-CRISPR/Cas13a reaction. A pod-shaped vest-pocket analysis device achieves the whole process for reaction execution, signal acquisition, and result interpretation. SCOPE can detect as low as 0.5 copies/µL (2.5 copies/reaction) of MPXV within 15 min from the sample input to the answer. We validate the developed assay on 102 clinical samples from male patients / volunteers, and the testing results are 100% concordant with the real-time PCR. SCOPE achieves a single-molecular level sensitivity in minutes with a simplified procedure performed on a miniaturized wireless device, which is expected to spur substantial progress to enable the practice application of CRISPR-based diagnostics techniques in a point-of-care setting.

Evaluation of analytical performance of the STANDARDTM M10 MPX/OPX assay for the simultaneous DNA detection and clade attribution of Monkeypox virus.

Monkeypox virus (MPXV) infection confirmation needs reliable polymerase chain reaction (PCR) assays; in addition, viral clade attribution is a key factor in containment measures, considering a more severe syndrome in clade I and the possibility of simultaneous circulation. This study evaluates the performance of all-in-one STANDARD M10 MPX/OPX (SD BIOSENSOR, South Korea - M10). Frozen samples from 205 subjects were selected and stratified according to routine test results (RealStar® Orthopoxvirus PCR Kit 1.0, Altona DIAGNOTICS, Germany - RS; RS-1): in detail, 100 negative skin lesions (SL) and 200 positive samples at the variable stage of infection were analysed. Positive samples were retested with RS (RS-2). Positive and Negative Percent Agreements (PPA, NPA) were calculated. The median (IQR) Ct values of RS and M10 (OPXV target) assays were highly similar. The PPA of M10 compared to RS-1 was 89.5% considering system interpretation, and 96.0% when the operator classified results as positive if any target was detected; NPA was 100%. Comparing the RS-2 run and M10, an overall concordance of 95.3% between assays was found; however, considering operator interpretation, M10 returned more positive results than RS-2. The occurrence of False-Negative results was likely associated with the influence of thawing on low viral concentration; no False-Positive tests were observed. All samples collected at the time of Mpox diagnosis were positive and M10 correctly attributed the clade (West-Africa/II). The M10 MPX/OPX assay demonstrated high reliability in confirming MPXV infection and clade attribution.

Mpox Diagnosis, Behavioral Risk Modification, and Vaccination Uptake among Gay, Bisexual, and Other Men Who Have Sex with Men, United Kingdom, 2022.

During the 2022 multicountry mpox outbreak, the United Kingdom identified cases beginning in May. UK cases increased in June, peaked in July, then rapidly declined after September 2022. Public health responses included community-supported messaging and targeted mpox vaccination among eligible gay, bisexual, and other men who have sex with men (GBMSM). Using data from an online survey of GBMSM during November-December 2022, we examined self-reported mpox diagnoses, behavioral risk modification, and mpox vaccination offer and uptake. Among 1,333 participants, only 35 (2.6%) ever tested mpox-positive, but 707 (53%) reported behavior modification to avoid mpox. Among vaccine-eligible GBMSM, uptake was 69% (95% CI 65%-72%; 601/875) and was 92% (95% CI 89%-94%; 601/655) among those offered vaccine. GBMSM self-identifying as bisexual, reporting lower educational qualifications, or identifying as unemployed were less likely to be vaccinated. Equitable offer and provision of mpox vaccine are needed to minimize the risk for future outbreaks and mpox-related health inequalities.

Demographic and clinical characteristics of people diagnosed with active sexually transmitted infections among monkeypox cases in Brazil: the 2022 outbreak.

The worldwide monkeypox (mpox) outbreak in 2022 showed a high frequency of sexually transmitted infections (STI). A cross-sectional study was carried out using secondary data from the Brazilian official mpox surveillance systems. A total of 10,169 mpox cases were identified, with a median age of 32 years. Among them, 92.3% were male at birth and 57.5% were men who have sex with other men (MSM). Approximately 11% were diagnosed with STI, including 5.8% with syphilis and 2.5% with genital herpes. Individuals aged from 25 to 34 years, MSM, individuals with HIV-positive status, and those manifesting skin eruptions or penile edema were associated with STI. Laboratory investigation for mpox must be implemented as a priority in STI clinics (especially for MSM) to mitigate neglected cases, ensure appropriate treatments, and prevent misdiagnoses.

Data-driven recombination detection in viral genomes.

Recombination is a key molecular mechanism for the evolution and adaptation of viruses. The first recombinant SARS-CoV-2 genomes were recognized in 2021; as of today, more than ninety SARS-CoV-2 lineages are designated as recombinant. In the wake of the COVID-19 pandemic, several methods for detecting recombination in SARS-CoV-2 have been proposed; however, none could faithfully confirm manual analyses by experts in the field. We hereby present RecombinHunt, an original data-driven method for the identification of recombinant genomes, capable of recognizing recombinant SARS-CoV-2 genomes (or lineages) with one or two breakpoints with high accuracy and within reduced turn-around times. ReconbinHunt shows high specificity and sensitivity, compares favorably with other state-of-the-art methods, and faithfully confirms manual analyses by experts. RecombinHunt identifies recombinant viral genomes from the recent monkeypox epidemic in high concordance with manually curated analyses by experts, suggesting that our approach is robust and can be applied to any epidemic/pandemic virus.

Mpox outbreak in Rio de Janeiro, Brazil: A translational approach.

Mpox is a zoonotic disease historically reported in Africa. Since 2003, limited outbreaks have occurred outside Africa. In 2022, the global spread of cases with sustained interhuman transmission and unusual disease features raised public health concerns. We explore the mpox outbreak in Rio de Janeiro (RJ) state, Brazil, in an observational study of mpox-suspected cases from June to December 2022. Data collection relied on a public healthcare notification form. Diagnosis was determined by MPXV-PCR. In 46 confirmed cases, anti-OPXV IgG was determined by ELISA, and seven MPXV genomes were sequenced. A total of 3095 cases were included, 816 (26.3%) with positive MPXV-PCR results. Most positive cases were men in their 30 s and MSM. A total of 285 (34.9%) MPXV-PCR+ patients live with HIV. Eight were coinfected with varicella-zoster virus. Anogenital lesions and adenomegaly were associated with the diagnosis of mpox. Females and individuals under 18 represented 9.4% and 5.4% of all confirmed cases, respectively, showing higher PCR cycle threshold (Ct) values and fewer anogenital lesions compared to adult men. Anti-OPXV IgG was detected in 29/46 (63.0%) patients. All analyzed sequences belonged to clade IIb. In RJ state, mpox presented a diverse clinical picture, represented mainly by mild cases with low complication rates and prominent genital involvement. The incidence in females and children was higher than usually reported. The observation of a bimodal distribution of Ct values, with few positive results, may suggest the need to review the diagnostic criteria in these groups.

INSaFLU-TELEVIR: an open web-based bioinformatics suite for viral metagenomic detection and routine genomic surveillance.

BACKGROUND: Implementation of clinical metagenomics and pathogen genomic surveillance can be particularly challenging due to the lack of bioinformatics tools and/or expertise. In order to face this challenge, we have previously developed INSaFLU, a free web-based bioinformatics platform for virus next-generation sequencing data analysis. Here, we considerably expanded its genomic surveillance component and developed a new module (TELEVIR) for metagenomic virus identification. RESULTS: The routine genomic surveillance component was strengthened with new workflows and functionalities, including (i) a reference-based genome assembly pipeline for Oxford Nanopore technologies (ONT) data; (ii) automated SARS-CoV-2 lineage classification; (iii) Nextclade analysis; (iv) Nextstrain phylogeographic and temporal analysis (SARS-CoV-2, human and avian influenza, monkeypox, respiratory syncytial virus (RSV A/B), as well as a "generic" build for other viruses); and (v) algn2pheno for screening mutations of interest. Both INSaFLU pipelines for reference-based consensus generation (Illumina and ONT) were benchmarked against commonly used command line bioinformatics workflows for SARS-CoV-2, and an INSaFLU snakemake version was released. In parallel, a new module (TELEVIR) for virus detection was developed, after extensive benchmarking of state-of-the-art metagenomics software and following up-to-date recommendations and practices in the field. TELEVIR allows running complex workflows, covering several combinations of steps (e.g., with/without viral enrichment or host depletion), classification software (e.g., Kaiju, Kraken2, Centrifuge, FastViromeExplorer), and databases (RefSeq viral genome, Virosaurus, etc.), while culminating in user- and diagnosis-oriented reports. Finally, to potentiate real-time virus detection during ONT runs, we developed findONTime, a tool aimed at reducing costs and the time between sample reception and diagnosis. CONCLUSIONS: The accessibility, versatility, and functionality of INSaFLU-TELEVIR are expected to supply public and animal health laboratories and researchers with a user-oriented and pan-viral bioinformatics framework that promotes a strengthened and timely viral metagenomic detection and routine genomics surveillance. INSaFLU-TELEVIR is compatible with Illumina, Ion Torrent, and ONT data and is freely available at https://insaflu.insa.pt/ (online tool) and https://github.com/INSaFLU (code).

Perichondritis and auricular cellulitis related to piercings as first manifestation of monkeypox / Pericondritis y celulitis auricular relacionadas con piercings como primera manifestación de viruela del mono

Introduction: Pinna infections are usually due to Staphylococcus aureus infection. It is common for the patient to have had an earring in the area of infection. Monkeypox infection has gone from being an endemic infection to a worldwide health emergency. Case summary: In this article we present five cases of monkeypox earring infection of the pinna and what common features we have seen that differentiate them from Staphylococcus aureus infection. Discussion: Symptoms of monkeypox include general malaise, fever with uni- or bilateral lymphadenopathy, and then the appearance within one or two days of skin lesions, we want to alert he otolaryngologist and the medical society to the possibility the diagnostic possibility of monkeypox in patients with an auricular perichondritis.(AU)

Introducción: Las infecciones del pabellón auricular se deben habitualmente a la infección por Staphilococcus Aureus. Es habitual que el paciente se haya realizado un pendiente en la zona de la infección. La infección por viruela del Mono ha pasado de ser una infección endémica a una emergencia sanitaria a nivel mundial. Caso: Exponemos en este artículo cinco casos de infección del pabellón auricular por pendiente por viruela del mono y que características comunes hemos visto que las diferencian de la infección por Staphilococcus Aureus. Discusión:Los síntomas de la viruela del mono incluyen malestar general, fiebre con linfadenopatía uni o bilateral, y posteriormente la aparición en uno o dos días de lesiones cutáneas, queremos alertar al otorrinolaringólogo y a la sociedad médica de la posibilidad diagnóstica de viruela del mono en pacientes con una pericondritis auricular.(AU)

Monkeypox: What the Dermatologist Needs to Know.

Monkeypox, an endemic disease in some African countries, has provoked public health activeness on a global scale that even the World Health Organization (WHO), invoking international health regulations, declared it a public health emergency of international concern (PHEIC). The WHO called attention of member states to exert maximum surveillance over the disease, its patients, and contact persons in order to standardize control measures. A need was directed to provide complete knowledge about the disease, allowing the administration of prior diagnoses as well as isolation and more effective epidemiologic control measures. An updated review on monkeypox emphasized upon having the fundamental aspects of the lesions caused by the disease, and appropriate management of patients upon clinical and epidemiologic evaluation.

Evaluation of 5 Polymerase Chain Reaction Assays for the Detection of Mpox Virus.

BACKGROUND: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak. METHODS: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay. RESULTS: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb. CONCLUSIONS: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains.

A high-throughput DNA analysis method based on isothermal amplification on a suspension microarray for detecting mpox virus and viruses with comparable symptoms.

BACKGROUND: Mpox is a zoonotic disease caused by mpox virus (MPXV) infection. Since May 2022, there has been a marked increase in human mpox cases in different regions. Rash, fever, and sore throat are typical signs of mpox. However, other viruses, such as the B virus (BV), herpes simplex virus types 1 (HSV-1), herpes simplex virus types 2 (HSV-2), and varicella zoster virus (VZV), can also infect people and cause comparable symptoms. Therefore, clinical symptoms and signs alone make distinguishing MPXV from these viruses difficult. RESULTS: In this study, we combined suspension microarray technology with recombinase-aided amplification technology (RAA) to establish a high-throughput, sensitive, and quantitative method for detecting MPXV and other viruses that can cause similar symptoms. The experimental results confirmed that the technique has outstanding sensitivity, with a minimum detection limit (LOD) of 0.1 fM and a linear range of 0.3 fM to 20 pM, spanning five orders of magnitude. The approach also exhibits exquisite selectivity, as the amplified signal can only be detected when the target virus nucleic acid is present. Additionally, serum recoveries ranging from 80.52% to 119.09% suggest that the detection outcomes are generally considered reliable. Moreover, the time required for detection using this high-throughput method is very short. After DNA extraction, the detection signal amplified by isothermal amplification on the bead array can be obtained in just 1 h. SIGNIFICANCE AND NOVELTY: Our research introduces a new technique that utilizes suspension microarray technology and isothermal amplification to create a high-throughput nucleic acid assay. This innovative method offers multiple benefits compared to current techniques, such as being cost-effective, time-efficient, highly sensitive, and having high throughput capabilities. Furthermore, the assay is applicable not only for detecting MPXV and viruses with similar symptoms, but also for clinical diagnostics, food safety, and environmental monitoring, rendering it an effective tool for screening harmful microorganisms.

Fast and Ultrasensitive Detection of Monkeypox by a Pyrococcus furiosus Argonaute System Coupled with a Short Amplification.

Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Pyrococcus furiosus Argonaute (PfAgo) is an artificial DNA-guided restriction cleavage enzyme programmable with 5'-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a PfAgo-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5'-phosphorylation guide DNAs were designed to guide PfAgo to cleave the amplicon to obtain an 18 bp 5'-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided PfAgo to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the PfAgo-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the PfAgo-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system's good performance. The PfAgo platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.

Performance of the Xpert™ Mpox PCR assay with oropharyngeal, anorectal, and cutaneous lesion swab specimens.

Anorectal and oropharyngeal exposures are implicated in sexual transmission of mpox, but authorized assays in the United States are only validated with cutaneous lesion swabs. Diagnostic assays for anorectal and oropharyngeal swabs are needed to address potential future outbreaks. The Cepheid Xpert® Mpox is the first point-of-care assay to receive FDA emergency use authorization in the United States and would be a valuable tool for evaluating these sample types. Our exploratory study demonstrates 100 % positive agreement with our in-house PCR assay for natural positive anorectal and oropharyngeal specimens and 92 % sensitivity with low-positive spiked specimens. The Xpert® assay detected viral DNA in specimens not detected by our reference PCR assay from four participants with mpox DNA at other sites, suggesting it may be more sensitive at low viral loads. In conclusion, the validation of the Xpert® for oropharyngeal and anorectal sample types can be rapidly achieved if clinical need returns and prospective samples become available.

Human monkeypox virus: Epidemiologic review and research progress in diagnosis and treatment.

Monkeypox virus (MPXV) is responsible for causing a zoonotic disease called monkeypox (mpox), which sporadically infects humans in West and Central Africa. It first infected humans in 1970 and, along with the variola virus, belongs to the genus Orthopoxvirus in the poxvirus family. Since the World Health Organization declared the MPXV outbreak a "Public Health Emergency of International Concern" on July 23, 2022, the number of infected patients has increased dramatically. To control this epidemic and address this previously neglected disease, MPXV needs to be better understood and reevaluated. In this review, we cover recent research on MPXV, including its genomic and pathogenic characteristics, transmission, mutations and mechanisms, clinical characteristics, epidemiology, laboratory diagnosis, and treatment measures, as well as prevention of MPXV infection in light of the 2022 and 2023 global outbreaks. The 2022 MPXV outbreak has been primarily associated with close intimate contact, including sexual activity, with most cases diagnosed among men who have sex with men. The incubation period of MPXV infection usually lasts from 6 to 13 days, and symptoms include fever, muscle pains, headache, swollen lymph nodes, and a characteristic painful rash, including several stages, such as macules, papules, blisters, pustules, scabs, and scab shedding involving the genitals and anus. Polymerase chain reaction (PCR) is usually used to detect MPXV in skin lesion material. Treatment includes supportive care, antivirals, and intravenous vaccinia immune globulin. Smallpox vaccines have been designed with four givens emergency approval for use against MPXV infection.

Co-Circulating Monkeypox and Swinepox Viruses, Democratic Republic of the Congo, 2022.

In September 2022, deaths of pigs manifesting pox-like lesions caused by swinepox virus were reported in Tshuapa Province, Democratic Republic of the Congo. Two human mpox cases were found concurrently in the surrounding community. Specific diagnostics and robust sequencing are needed to characterize multiple poxviruses and prevent potential poxvirus transmission.

Dynamics of Mpox infection in Nigeria: a systematic review and meta-analysis.

The seasonal outbreaks of Mpox continue in most parts of West and Central Africa. In the past year, Nigeria had the highest number of reported cases. Here, we used the PRISMA guidelines to carry out a systematic review and meta-analysis of available evidence on Mpox in Nigeria to assess the prevalence, transmission pattern, diagnostic approach, and other associated factors useful for mitigating the transmission of the disease. All relevant observational studies in PubMed/MEDLINE, Embase, AJOL, Web of Science, Scopus and Google Scholar on Mpox in Nigeria were assessed within the last fifty years (1972 to 2022). In all, 92 relevant articles were retrieved, out of which 23 were included in the final qualitative analysis. Notably, most of the cases of Mpox in Nigeria were from the southern part of the country. Our findings showed a progressive spread from the southern to the northern region of the country. We identified the following factors as important in the transmission of Mpox in Nigeria; poverty, lack of basic healthcare facilities, and risk of exposure through unsafe sexual practices. Our findings reiterate the need to strengthen and expand existing efforts as well as establish robust multi-sectoral collaboration to understand the dynamics of Mpox Nigeria.